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CELL SCIENCE AT A GLANCE

Membrane curvature at a glance

Harvey T. McMahon1,* and Emmanuel Boucrot2,*

ABSTRACT

Membrane curvature is an important parameter in defining the

morphology of cells, organelles and local membrane subdomains.

Transport intermediates have simpler shapes, being either spheres

or tubules. The generation and maintenance of curvature is of central

importance for maintaining trafficking and cellular functions. It is

possible that local shapes in complex membranes could help to

define local subregions. In this Cell Science at a Glance article and

accompanying poster, we summarize how generating, sensing and

maintaining high local membrane curvature is an active process that

is mediated and controlled by specialized proteins using general

mechanisms: (i) changes in lipid composition and asymmetry, (ii)

partitioning of shaped transmembrane domains of integral membrane

proteins or protein or domain crowding, (iii) reversible insertion of

hydrophobic proteinmotifs, (iv) nanoscopic scaffolding by oligomerized

hydrophilic protein domains and, finally, (v) macroscopic scaffolding

by the cytoskeleton with forces generated by polymerization and by

molecular motors. We also summarize some of the discoveries about

the functions of membrane curvature, where in addition to providing

cell or organelle shape, local curvature can affect processes like

membrane scission and fusion as well as protein concentration and

enzyme activation on membranes.

KEY WORDS: Amphipatic helix, BAR domain, Bilayer asymmetry,

Lipids, Membrane curvature

1MRC Laboratory of Molecular Biology, Francis Crick Avenue, CambridgeCB2 0QH, UK. 2Institute of Structural and Molecular Biology, University CollegeLondon & Birkbeck College, London WC1E 6BT, UK.

*Authors for correspondence (hmm@mrc-lmb.cam.ac.uk; e.boucrot@ucl.ac.uk)

� 2015. Published by The Company of Biologists Ltd | Journal of Cell Science (2015) 128, 1065–1070 doi:10.1242/jcs.114454

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IntroductionThe formation of many intracellular membrane compartments

allows the cell to compartmentalize proteins, thereby supportingthe complex and timely coordination of the thousands ofbiochemical reactions required for eukaryotic life. Becausemembranes are constantly turned over by trafficking

intermediates (vesicles or tubules), the ability to generate high-curvature compartments is necessary for eukaryotic life. Lipidbilayers forming eukaryotic cell membranes have elastic

properties that make them resistant to spontaneous bending and,thus, active mechanisms are required to shape them; these havebeen conserved throughout evolution. Many proteins contain

domains or motifs that are specialized in sensing, generating orstabilizing membrane curvature. In addition, there are proteinsthat act directly by changing lipids, whereas others provide

scaffolding and forces that impose tension on membranes.

Mechanisms of membrane curvature generationand stabilizationChange in lipid composition and asymmetryLipids have intrinsic shapes depending on the size of theirheadgroups and their acyl chain compositions (length and

saturation). These features determine their side-by-side packing,thereby imposing a shape on the monolayer. If many lipids withsimilar shape cluster together, then a monolayer will adopt the

spontaneous curvature of the local lipids. Owing to bilayercoupling, the whole membrane might also change shape (seeposter) (McMahon and Gallop, 2005; Cooke and Deserno, 2006).

Thus a bilayer will adopt a spontaneous curvature equal to thedifference between the spontaneous curvatures of its inner andouter monolayers, which are themselves dictated by the weightedaverage of the spontaneous curvature of their constituent lipids

(Kozlov et al., 1992; Leikin et al., 1996).Phosphatidylcholine (PtdCho) and phosphatidylserine (PtdSer)

are cylindrical lipids that form a flat monolayer. Lipids such as

phosphatidylethanolamine (PtdEtn), phosphatic acid, diacylglycerol(DAG) or cardiolipin, which have a smaller polar headgroup thanthat of PtdCho, have a roughly conical shape and thus impose a

negative curvature, where a monolayer with such lipids bendsin such a fashion that the headgroups come closer together.Conversely, a large headgroup to acyl chain ratio, such as inlysophosphatidylcholine (LPC) or the large headgroups in

phosphatidylinositol phosphates (PtdIns) confers an invertedconical shape to the lipids, thereby favoring the bending of themembrane into a positive curvature, bending the monolayer away

from the headgroups (reviewed in Chernomordik and Kozlov, 2003;Di Paolo and De Camilli, 2006; Zimmerberg and Kozlov, 2006).

Acyl chain saturation also influences lipid geometry; any

double bond (such as in oleic acid) induces a kink in the chainand thus forces it to occupy more space than its saturatedcounterpart. Thus, the ratio between the size of the polar

headgroup and acyl chain saturation defines the overallgeometry of the lipids (Vanni et al., 2014; Pinot et al., 2014).The artificial combination of a bulky headgroup andmonounsaturation of both acyl chains, such as in the artificial

lipids 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS) or Ni2+-NTA-DOGS, favors spontaneous curvature (Bigay and Antonny,2012). Conversely, sterols increase the packing of cylindrical

sphingolipids (such as sphingomyelin) and of PtdCho, and thusdisfavor spontaneous curvature (Ikonen, 2008).

Active maintenance of lipid asymmetry by type IV P-type

ATPases (lipid flippases) that translocate phospholipids (e.g.

PtdSer or PtdEtn) directionally from the extracellular leaflet to thecytosolic face (Daleke, 2007; Devaux et al., 2008) supports changes

in membrane curvature and vesicular budding by generating adifference in the partitioning of lipids between the two leaflets(Poulsen et al., 2008; Ruaud et al., 2009; Xu et al., 2009). Changesin the lipid composition of one monolayer by lysophospholipid

acyltransferases, phospholipase A or sphingomyelinases alsoinduce local membrane curvature (reviewed in Graham andKozlov, 2010). Finally, the clustering of certain lipids by

bacterial toxins, such as cholera toxin B or Shiga toxin B, favorsthe concentration of lipids with the same shape (conical, invertedconical or cylindrical; see poster) on one leaflet and coincides with

the generation of negative membrane curvature (Ewers et al., 2010;Romer et al., 2007).

Partitioning of shaped transmembrane domains and protein crowdingTransmembrane proteins (ion channels, transporters andreceptors) that have an intrinsic conical or inverted conicalshape can bend their associated membranes (Aimon et al., 2014;

Fertuck and Salpeter, 1974; Fribourg et al., 2014; MacKinnon,2003; Unwin, 2005). The asymmetry of extramembrane domainsof transmembrane proteins might also result in bending. Thus,

receptors might cause membrane bending by protein crowdingwhere the membrane bends away from the side with the largestdomains (Copic et al., 2012). In addition, many integral

membrane proteins oligomerize – either directly amongthemselves or through connecting proteins (Boudin et al., 2000;Eckler et al., 2005), which results in local scaffolding of

membranes. The clustering of transmembrane receptors, such astransferrin or low-density lipoprotein (LDL) receptors, intoforming endocytic pits supports the generation of membranecurvature by stabilizing forming clathrin-coated pits (Ehrlich

et al., 2004). These mechanisms might play a role in sortingreceptors into (or excluding them from) membrane traffickingcarriers. A high local concentration of proteins that bind to the

membrane surface has been suggested to induce membranecurvature by a crowding mechanism (Stachowiak et al., 2012),although the contribution of this mechanism is still unclear

(recently discussed in Kozlov et al., 2014).

Insertion of hydrophobic protein motifsInsertion of a small hydrophobic protein motif in between the

lipid headgroups is a very efficient way of inducing localmembrane curvature (see poster). It acts as a ‘wedge’; given thatthe shallow insertion is in the outer part of the membrane, this

means that the apex of the wedge is in the center of the bilayerand, thus, the radius of curvature is small (the thickness of themonolayer) (Ford et al., 2002). If there is a single insertion, the

bending of the membrane is dissipated rapidly in space, but ifseveral insertions are held in close proximity (within the sameprotein or within complex protein assemblies), the resulting

membrane curvature is much more pronounced (Campelo et al.,2008; McMahon et al., 2010). Amphipathic helices are presentin many proteins that are involved in membrane remodelingthroughout the entire cell, such as epsin, endophilin,

amphiphysin, Bin2, nadrin (also known as ARHGAP17), Arfproteins, Sar1 and atlastin GTPases, GRK5, ArfGAP1, ArfGEFGBF1, GMAP-210 (also known as TRIP11), NUP133, barkor, a-

synuclein, annexin B12, Atg3 and the endosomal sortingcomplexes required for transport (ESCRT)-III protein CHMP4B(reviewed in Drin and Antonny, 2010). Amphipathic helices vary

in lengths and in their affinities for charged lipids (dictated by the

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nature of the amino acids on the edges of the hydrophobic face ofthe helix), and thus have varying abilities to sense or induce

membrane curvature. Several bacterial proteins, such as DivIVAor SpoVM, use amphipathic helices for their subcellulartargeting, suggesting that curvature sensing is a very ancientmechanism (Lenarcic et al., 2009; Ramamurthi et al., 2009).

The insertion of hydrophobic loops that are present at the tip ofthe C2 domains of synaptotagmin-1 and Doc2b (Groffen et al.,2010; Hui et al., 2009; Martens et al., 2007) or at the membrane-

binding interface of Pacsin and EHD2 also induce membranecurvature by acting as wedges (Daumke et al., 2007; Plomannet al., 2010); although, in the latter case, protein oligomerization

and scaffolding also contribute to the generation of curvature.Consistent with their extensive insertion into the lipid bilayer, C2domains induce a high degree of curvature (McMahon et al.,

2010). As C2 domains bind to membrane in a Ca2+-dependentmanner, their membrane bending activity is tightly regulated (Huiet al., 2009; Martens et al., 2007).

Nanoscopic scaffolding by oligomerized hydrophilic protein domainsScaffolding by peripheral proteins supports membrane curvatureat a nanoscopic level (curvature on the scale of protein domains),

whereby the shape of the membrane-binding interface is imposedon the membrane. Nanoscopic proteins might also assemble intolarger structures that have a shape that is formed by the oligomer

and thus affect membrane curvature at the microscopic level.

Protein oligomerizationCoat proteins such as clathrin, COPI and COPII are key factorsthat stabilize membrane curvature during vesicle budding (Jensenand Schekman, 2011; Kirchhausen, 2000; McMahon andBoucrot, 2011; Zanetti et al., 2012). They do not bind to

membrane directly and rely on adaptor proteins to link them tomembranes (see poster). The capacity of the coat proteins to bendto membranes depends on the rigidity of the assembled

polyhedral coat and the transmission of this shape to themembrane (Copic et al., 2012). Members of the dynaminGTPase superfamily induce membrane curvature by self-

polymerizing into spirals (reviewed in Faelber et al., 2012;Ferguson and De Camilli, 2012; Morlot and Roux, 2013; Praefckeand McMahon, 2004). However, dynamin requires a pre-existingcurvature to assemble efficiently, at least in the presence of

membrane tension. Proteins such as caveolin, flotillins or thereticulons oligomerize (directly or through bridging proteins) aswell as insert into membranes, thereby facilitating the formation

or stabilization of curvature at caveolae and the endoplasmicreticulum (ER), respectively (Hu et al., 2011; Parton and delPozo, 2013; Shibata et al., 2009).

BAR domainsBAR (Bin/Amphiphysin/Rvs) domains are crescent-shaped

dimeric modules that bind to membranes. The prototypicalBAR domain protein is amphiphysin, which binds to membranethrough its concave face (Peter et al., 2004). The initial binding ismediated by electrostatic interaction between positively charged

amino acids (lysine or arginine) of the BAR module andnegatively charged lipids [PtdSer and/or phosphatidylinositolphosphates, such as PtdIns(4,5)P2] (Mim and Unger, 2012; Rao

and Haucke, 2011). Some BAR domains can further oligomeriseby forming ‘tip-to-tip’ or ‘side-to-side’ interactions, therebyincreasing their local concentration, which makes it more likely

that they can impose their intrinsic curvature locally on the

membrane (Mim and Unger, 2012; Rao and Haucke, 2011). BARdomains favor the formation of membrane tubules and disfavor

the more extreme curvature needed for membrane scission (Peteret al., 2004; Boucrot et al., 2012).

Macroscopic scaffolding by the cytoskeletonScaffolding by the actin, intermediate filament and microtubulecytoskeleton supports cell membrane curvature at themacroscopic scale (in the range of multiple microns in radius),

which is present in large intracellular organelles, such as theGolgi or the ER. The cytoskeleton also mediates the macroscopiccurvature of the plasma membrane in filopodia and lamellipodia,

or of the specific arrangement of the plasma membrane duringcell division (i.e. cortical actin supporting mitotic cell rounding),phagocytosis (membrane ruffling) or in specialized cellular

shapes (neurons or visual cones) (Rohn and Baum, 2010;Sheetz, 2001). The cytoskeleton maintains cell membranetension by connecting to the bilayer at regular intervals, and itimposes macroscopic shapes by providing an underlying

scaffold (Doherty and McMahon, 2008). Active membranepulling or pushing by kinesins, dynein and myosin also inducesconsiderable membrane reorganization and supports some of the

organelle morphologies (e.g. ER, Golgi and endosomes)(reviewed in Leduc et al., 2010).

Synergistic action of multiple mechanismsA combination of several of the aforementioned mechanismsusually takes place to efficiently induce membrane sculpting.

Proteins with insertion motifs, scaffolding abilities or both bind tonumerous other proteins that are involved in the same cellularprocess and, thus, form a network of interactions that combinesensing, induction and stabilization of curvature (McMahon and

Gallop, 2005). For example, during the formation of a clathrin-coated pit, several of the membrane-bending mechanismspresented above take place. The asymmetrical partitioning of

inverted conical lipids [PtdSer and PtdIns(4,5)P2] between thetwo membrane leaflets and their clustering by several proteins –some of which have, in addition, membrane inserting (epsin) or

scaffolding modules (amphiphysin) – all occur within anoligomeric protein scaffold that is being held together by theclathrin coat (McMahon and Boucrot, 2011).

Functions of membrane curvatureThe control of biological membrane shapes is central toeukaryotic life. Thus, the formation of high-curvature transport

intermediates is necessary for compartmentalization, and thecharacteristic shapes of organelles are likely to be related to theirfunction; indeed, the ability to control fission and fusion, local

tethering or enzyme reactions might also be shape dependent (seeposter; reviewed in McMahon and Gallop, 2005; Shibata et al.,2009).

Organelle shapingMost intracellular organelles have characteristic membraneshapes that make them easily recognizable by their morphology

in electron micrographs. These shapes are mediated by the extentand localization of membrane curvature, which give rise to thetypical shapes of the ER, the Golgi, mitochondria, lipid droplets,

autophagosomes or endosomal and secretory carriers (Shibataet al., 2009). However, these characteristic shapes are dynamic,and they change according to the needs of the cell (e.g.

mitochondrial fission or fusion) or the maturation of the

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organelle (e.g. formation or consumption of endocytic or synapticvesicles) (Friedman and Nunnari, 2014; Haucke et al., 2011;

Rafelski and Marshall, 2008).

Membrane scissionScission between two membranes requires the localized and

timely connection in trans of two bilayer membranes to separatean organelle from its membrane of origin, while avoidingleakiness. Membrane scission is a key step in the budding of

coated vesicles from the ER (COPII coat), Golgi (COPI coat),trans-Golgi network (AP-1 and AP-3 clathrin coats), endosomes(ESCRT complexes) and plasma membranes (AP-2 clathrin coat),

as well as in virus budding (Ferguson and De Camilli, 2012;Hurley and Hanson, 2010; Rossman and Lamb, 2013). Membersof the dynamin superfamily are the main drivers of membrane

scission. They oligomerize at the neck of nascent vesicles and,upon hydrolysis of GTP, they trigger fusion between the two lipidbilayers, thereby causing the scission of the neck and detachmentof the vesicle (Ferguson and De Camilli, 2012; Praefcke and

McMahon, 2004). High local concentrations of hydrophobicinsertions at the neck of constricted vesicle carriers supports thefission of nascent membrane-trafficking vesicles by creating

sufficient stress on the bilayer to favor scission, as it is the casefor Sar1 at COPII vesicles (Lee et al., 2005) and epsin, which cansupport dynamin at clathrin-coated pits (Boucrot et al., 2012).

This mechanism is also likely to have a role in virus and exosomebudding, as well as in the formation of multivesicular bodies(MVBs) in endosomes, because key proteins involved in these

processes have amphipathic helices (Buchkovich et al., 2013;Rossman et al., 2010).

Membrane targeting and tetheringMembrane curvature sensing has an important role in vesicletargeting, which is exerted through tethering proteins such as theGolgin GMAP-210 that acts at the cis Golgi (Drin et al., 2007;

Drin et al., 2008). This protein uses amphipathic helices at the tipof long molecular strings to sense high curvature on incomingvesicles before the engagement of soluble NSF attachment

protein receptors (SNAREs), thereby increasing the fidelity ofvesicle targeting. Other examples are the highly curvedmembrane sensing by the autophagosome-specific adaptorbarkor (also known as Atg14L) that mediates the subsequent

recruitment of class III phosphatidylinositol 3-kinase duringautophagosome biogenesis (Fan et al., 2011), and the synergybetween cargo recruitment by the autophagy receptor Atg19 and

membrane bending of Atg8-coated membranes duringautophagophore wrapping around the cargo (Sawa-Makarskaet al., 2014).

Membrane fusionThe fusion between a vesicle and its target membrane is mediated

by the assembly of cognate SNARE complexes that bring thetwo apposing membranes into close proximity upon assembly(see poster) (Jahn and Scheller, 2006; Martens and McMahon,2008). However, SNARE complexes might be aided by both

the lipid composition of the membrane and the coordinatedaction of membrane-bending modules, such as the C2 domainsof synaptotagmin and Doc2 proteins, or the amphipathic helix

of complexin, which facilitate and accelerate membrane fusionand neurotransmitter release at neuronal synapses (Groffenet al., 2010; Hui et al., 2009; Martens et al., 2007; Snead et al.,

2014).

Protein sortingThe intrinsic shapes of transmembrane receptors might mediate

their partitioning into regions of the membrane that accommodatethe relevant shapes. This might be the case for nicotinicacetylcholine and glutamate receptors as well as Ca2+ or K+

channels that appear to concentrate at post-synaptic regions and

other areas of high membrane curvature (see poster) (Aimon et al.,2014; Fertuck and Salpeter, 1974; MacKinnon, 2003; Unwin,2005). In making vesicle trafficking intermediates, the initial bud

formation might help to concentrate proteins that favor the budcurvature or, alternatively, help to exclude proteins that either donot have an adaptor to localize them to the bud or have an intrinsic

curvature that forces them to leave the bud. The concentration ofhigh-curvature-sensing and -inducing proteins, such as Sar1, theESCRT complex or sorting nexin proteins, is also important for the

organization of ER exit sites or endosomal sorting and recycling(Cullen and Korswagen, 2012; Okamoto et al., 2012).

Enzyme and protein activationSeveral enzymes display curvature-sensitive activities. For

instance, the phosphoinositide phosphatase synaptojanin has ahigher activity on highly curved membrane (Chang-Ileto et al.,2011). The lipid-extraction capacity of the sterol transporter Osh4p

(also known as Kes1p) is also greater at a high membrane curvature(de Saint-Jean et al., 2011; Drin et al., 2007), as is lipidation of theautophagy protein LC3 (encoded by MAP1LC3A) or GABARAPby Atg3 (Nath et al., 2014). The small GTPases Arf, Arl and Sar1,

as well as some of their guanosine exchange factors (GEFs)and GTPase-activating proteins (GAPs) are also activated bymembrane curvature (Bigay et al., 2005; Krauss et al., 2008; Lee

et al., 2005; Lundmark et al., 2008). It is likely that future work willuncover many other instances of protein activation by membranecurvature.

Conclusions and perspectivesIn addition to the lipid properties that are inherent to the

membrane bilayer, several distinct mechanisms exist that allowproteins to sense, stabilize or generate a high local membranecurvature. Several of these mechanisms can be utilized in concert

during complex membrane remodeling processes, such asorganelle shaping and vesicle budding, scission and fusion.Consistent with its universality within eukaryotic cells, it is verylikely that additional mechanisms underlying membrane shaping

will be uncovered in the near future and that further cellularfunctions will be recognized to depend on membrane curvature.

Competing interestsThe authors declare no competing or financial interests.

FundingThis work was supported by a Medical Research Council UK grant [grant numberU105178805] to H.T.M.; and a Biotechnology and Biological Sciences ResearchCouncil David Phillips Research Fellowship [grant number BB/I018921]; and aprize from the Lister Institute for Preventive Medicine to E.B. Deposited in PMC forrelease after 6 months.

Cell science at a glanceA high-resolution version of the poster is available for downloading in the onlineversion of this article at jcs.biologists.org. Individual poster panels are availableas JPEG files at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.114454/-/DC1.

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